Biological Control 32 (2005) 155–163 www.elsevier.com/locate/ybcon Ecology of Metarhizium anisopliae in soilless potting media and the rhizosphere: implications for pest management Denny J. Bruck¤ USDA-ARS, Horticultural Crops Research Laboratory, 3420 NW Orchard Avenue, Corvallis, OR 97330, United States Received 8 July 2004; accepted 14 September 2004 Abstract Wholesale container-grown ornamentals are often maintained at the nursery for at least two growing seasons and are subject to infestation by black vine weevil (BVW), Otiorhynchus sulcatus F., for several months each year. Therefore, a potting media amend- ment aimed at controlling BVW needs to persist for an extended period of time. These studies were conducted to determine the per- sistence and ecology of Metarhizium anisopliae (MetchnikoV) Sorokin incorporated into peat and bark-based potting media with and without a crab meal amendment in container-grown Picea abies ‘Nidiformis.’ Rooted cuttings of P. abies were planted into pot- ting media amended with M. anisopliae (1 g of formulated product/L; »6 log10 CFU/g dry potting media). The fungal population in bulk potting media was quantitatively determined using selective media at 14, 21, 28, 35, 49, 63, 77, 91, 105, 119, 143, 175, 203, 231, 258, 287 and 342 days. The fungal population in the rhizosphere was quantitatively determined at 203, 231, 258, 287, and 342 days. M. anisopliae colonized the rhizosphere of P. abies and the fungal population in the rhizosphere was signiWcantly greater than in the surrounding bulk media. M. anisopliae persisted in the peat and bark-based potting media at 6.22 and 5.74 log10 CFU/g dry potting media for 342 days, respectively. Bioassays using bark and peat-based potting media inoculated with M. anisopliae at 6 log10 CFU/g dry potting media resulted in 93.5% and 97.5% infection of last instar BVW, respectively. P. abies roots inoculated with M. anisopliae infected 76% of 2nd–3rd instar BVW. Inoculation of roots with M. anisopliae represents a novel method for delivering entomopath- ogenic fungi and would greatly reduce application costs. Factors associated with fungal biology outside the host may be more impor- tant than virulence in a laboratory bioassay. 2004 Elsevier Inc. All rights reserved. Keywords: Metarhizium anisopliae; Otiorhynchus sulcatus; Fungal biology; Rhizosphere colonization 1. Introduction the environment in the absence of their host in hopes that they will persist and infect their target once the host The inconsistent performance of biological control immigrates into the treated area. One system utilizing agents is often associated with an incomplete under- such an approach is the use of Metarhizium anisopliae standing of the ecological constraints of the biological (MetchnikoV) Sorokin (Hypocreales: Clavicipitaceae) to system in which they are placed. This is particularly true control the black vine weevil (BVW), Otiorhynchus sulc- for entomopathogenic fungi. There is little or no knowl- atus F., (Coleoptera: Curculionidae). The BVW is a edge of their biology outside of their insect host. How- polyphagous insect that is a severe pest of Weld-and con- ever, these fungi are often inundatively introduced into tainer-grown ornamentals as well as small fruit crops worldwide (Moorhouse et al., 1992). Economic losses are mostly associated with poor plant growth due to larval * Fax: +1 541 738 4025. root feeding and lost shipments due to quarantine issues. E-mail address: [email protected]. Other losses are associated with the cost of control, the 1049-9644/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2004.09.003 156 D.J. Bruck / Biological Control 32 (2005) 155–163 destructive sampling needed for larval scouting and cos- proliferate in the environment they are introduced (Nel- metic quality reduction due to leaf feeding (adult notch- son et al., 1994). Proliferation of rhizosphere competent ing of leaves). microbes for the biological control of soil borne plant Metarhizium anisopliae has been studied extensively diseases has been demonstrated using fungi (Harman, for the biological control of a wide range of insect pests, 1992) and bacteria (Kloepper and Beauchamp, 1992). including BVW (Booth and Shanks, 1998; Moorhouse For consistent use of microbes for biological control of et al., 1993a,b; Poprawski et al., 1985; Soares et al., 1983) plant diseases, it is essential that the biology and ecology and various other soil borne pests such as Popillia japon- of the biological control agent be understood completely ica (Coleoptera: Scarabaeidae) (Villani et al., 1994), (Lo et al., 1998). An understanding of the parameters Ligyrus subtropicus (Coleoptera: Scarabaeidae) (Raid impacting the ability of M. anisopliae to proliferate, and Cherry, 1992), Antitrogus parvulus (Coleoptera: maintain an eYcacious population and persist in the Scarabaeidae) (Samuels et al., 1990), Adoryphorus cou- microenvironment where control is desired would loni (Coleoptera: Scarabaeidae) (Rath, 1988) and Diab- improve biological control eYcacy. rotica undecimpunctata (Coleoptera: Chrysomelidae) All Wlamentous fungi with chitin as a major cell wall (Krueger and Roberts, 1997). The use, however, of M. component produce chitinases at all periods of active anisopliae and other entomopathogenic fungi for control growth (Gooday et al., 1992). Several insect pathogens of soil borne insects is often inconsistent due to limited including M. anisopliae, M. Xavoviride Gams and Rozsy- spore redistribution and persistence (Storey and Gard- opal (Hypocreales: Clavicipitaceae) and B. bassiana pro- ner, 1987, 1988; Storey et al., 1989). duce a complex mixture of chitinolytic enzymes during Increased understanding of the ecology of entomo- growth on insect cuticle (St. Leger et al., 1996). The pro- pathogens outside their host has enhanced our ability to duction of chitinase by M. anisopliae is dependent on eVectively utilize entomopathogens as biological control chitin availability (de Siqueria Pinto et al., 1997; St. agents. For example, Lewis and colleagues (Bing and Leger et al., 1996). M. anisopliae produces a heteroge- Lewis, 1991, 1992) observed that Beauveria bassiana (Bal- nous array of chitinases which may play a role in its abil- samo) Vuillemin (Hypocreales: Clavicipitaceae) grows ity to adapt to diVerent environments (St. Leger et al., endophytically within the green tissues of Zea mays L. 1993). (Cyperales: Poaceae). They then demonstrated that endo- The objectives of these studies were to quantify the phyte forming isolates of B. bassiana eVectively managed population of M. anisopliae over time in peat and bark- populations of the European corn borer, Ostrinia nubil- based potting media, determine the eVect of crab meal alis (Lepidoptera: Crambidae) (Lewis et al., 2002) while amendment (source of chitin) on the population size and being non-pathogenic to Z. mays (Lewis et al., 2001). persistence of M. anisopliae, determine if M. anisopliae Isolates of entomopathogenic fungi traditionally have colonized the rhizosphere of Picea abies (L.) Karst. been selected for development as biological control (Pinales: Pinaceae) ‘Nidiformis’ and determine if roots agents based on bioassay results from the laboratory inoculated with M. anisopliae were able to protect plants with little emphasis on understanding other fungal traits. from BVW larval feeding. Recently, an isolate of M. anisopliae (ARSEF 1080) was demonstrated to be rhizosphere competent (Hu and St. Leger, 2002). While the notion of fungal populations 2. Materials and methods increasing in the rhizosphere is not new, this was the Wrst report of an entomopathogenic fungus doing so. In Weld Two types of soilless potting media typically used in studies, the population of M. anisopliae in bulk soil container grown nursery production were used in the decreased from 105 to 103 propagules/g soil after several experiments. A 2:1 mixture of peat moss (Sunshine Mix months, while populations in the inner rhizosphere of #3, Sun Gro Horticulture, Bellevue, WA) and turkey grit cabbage plants remained at 105 propagules/g soil (Hu (Cherry Stone Grit #3, New Ulm, MN) and a bark-based and St. Leger, 2002). A signiWcantly larger fungal popu- media (OBC Northwest Nursery Mix #1, OBC North- lation in the inner rhizosphere compared to the outer west, Canby, OR) which consists of 70% Wne bark, 20% rhizosphere suggests that response to root exudates is mulch, 10% pumice. A commercial formulation of M. ani- involved in the rhizosphere eVect (Hu and St. Leger, sopliae (strain F52) [Earth BioSciences (New Haven, CT 2002) or that sporulation is enhanced in the rhizosphere. [formally Taensa Company])] was used. The formulated Root-deposited photosynthate is an important source of product consisted of M. anisopliae that had sporulated on readily available carbon for microbes in the rhizosphere rice grains and was then dried. The crab meal amendment (Butler et al., 2003). Rhizosphere competence is an attri- used was Eco-Logic (AgriGulf, Bayou La Batre, AL). bute of a microbe but the rate of colonization also The experiment was arranged as a randomized com- depends on the traits of the host (O’Connell et al., 1996). plete block in a split–split plot design with four replica- Biological control agents diVer fundamentally from tions. The whole plot was the type of soilless potting chemical agents in that in order to be eVective, they must media (peat, bark), the sub-plot the M. anisopliae D.J. Bruck / Biological Control 32 (2005) 155–163